The present invention, in some embodiments thereof, relates to methods of diagnosing and monitoring liver diseases and, more particularly, but not exclusively, to liver fibrosis.
Soluble secreted proteins that are expressed uniquely in specific organs, or proteins whose formation or secretion is regulated by disease states, are excellent markers for disease. The reason for this is that the disease can be diagnosed by simply measuring the level of the secreted protein in serum of a potential patient. The level of a secreted protein in serum can be easily measured in a number of different ways that are well known in the art, such as ELISA assay and Western blotting, directed at quantifying marker levels in the serum sample. However, in order to have a good marker for a disease, the secreted protein must have distinctly different levels in normal and disease tissues. Furthermore, in order to provide accurate diagnoses in diseases that must be caught at early development stages in order to enable efficient treatment, such as cancer or fibrosis, the marker must have distinct expression or secretion levels even at an early stage of disease development.
Liver function can be affected by many chemicals, medicines, diet regimes, environmental poisons, alcohol abuse and viral infections that lead to hepatitis. The most common complications are liver fibrosis and cirrhosis. Generally, the origins of liver fibrosis that leads in its advanced stages to cirrhosis are common complications of Hepatitis B and C.
Hepatitis B is very common in Africa and in Asia, especially in the Philippines and in China and is endemic in the Middle East. In Europe and North America the incidence of known carriers is about 1 in a 1000 people. Worldwide, it is estimated that there are over 350 million hepatitis B (HBV) carriers, which represents 5% of the world's population. In addition it is estimated that 10 to 30 million people are infected with the hepatitis B virus each year. 10% of the people infected with HBV develop chronic infection. People with a chronic HBV infection are at risk of liver damage and around 20-30% of these people later develop cirrhosis.
Hepatitis C (HCV) is almost as common, and it is estimated that there are approximately 200 million people worldwide infected with the virus. There are up to 230,000 new HCV infections every year in the U.S. alone. Currently, 8,000 to 10,000 people infected with HCV die each year. Over the next 10-20 years, chronic HCV is predicted to become a major burden on the health care system, as patients who are currently asymptomatic with a relatively mild form of the disease, progress to end-stage liver disease and develop hepatocellular carcinoma. Progressive hepatic fibrosis and cirrhosis develop in 20% to 30% of patients with chronic HCV. There is no vaccine but liver fibrosis caused by this virus can be treated at early stages. Predictions in the USA indicate that there will be a 60% increase in the incidence of cirrhosis, a 68% increase in hepatoma incidence, a 279% increment in incidence of hepatic decompensation, a 528% increase in the need for transplantation, and a 223% increase in liver death rate. Altogether the number of fibrotic and cirrhotic patients worldwide in need of periodic diagnosis can be estimated at around 20 million, with up to 2 million added each year. With regard to the number of pre-fibrotic patients that would benefit from an early diagnosis, there could be several hundred million worldwide.
Generally, blood tests for liver function are based on the level of several markers such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamic transpeptidase (GGT), bilirubin, albumin and prothrombin time (PT) in the serum. However, while the markers used in such “liver function tests” are capable of assessing hepatocyte integrity, which might be indicative of liver damage, most of them, except albumin and prothrombin, are not indicative of the synthesis function of the liver. Albumin, which is produced in the liver and circulates in the blood, is affected only when a liver disease is at a severe stage. On the other hand non-hepatic diseases such as nephrotic syndromes can affect albumin levels. Similarly, prothrombin, which is used to evaluate blood clotting disorders, is insensitive to mild liver disease and can be also affected by non-hepatic conditions such as dietary deficiencies or the use of anti-coagulants. Likewise, abnormal levels of bilirubin can result from hemolysis, ineffective erythropoiesis and other non-hepatic syndromes. In addition, as ALT, AST, ALP and GGT are also produced in organs other than the liver, their blood levels can be elevated in a wide range of non-hepatic diseases. Biochemical screening of healthy, asymptomatic people has revealed that up to 6% of the population exhibit abnormal levels of liver enzymes. However, the prevalence of liver disease in the general population is significantly lower (about 1%) (Gopal and Rosen, 2000, Postgraduate medicine; 107(2):100-2, 105-9, 113-4). Even though the current serum biochemical test pattern may suggest a specific diagnosis, confirmation usually requires further investigation using imaging studies and, possibly, liver biopsy.
Experimental serum markers that have been proposed for diagnosis of liver fibrosis include extracellular matrix (ECM) macromolecules and their degradation products such as N-terminal propeptide of type-III collagen (PIIINP), or the aminoterminal domain of procollagen type-IV (PIVNP), hyaluronic acid (HA), prolyl-hydroxylase, laminin, matrix metalloproteinase-1. The main problem with these markers is that they are not liver specific, not highly sensitive and can also reflect inflammation processes in other tissues. Recently, tests that combine several markers have been developed, for example Fibrotest (which includes α-2-macroglobulin, haptoglobin, apolipoprotein A1, GGT and total bilirubin). These and other combination tests provide a reasonable diagnosis for advanced fibrosis but are not satisfactory at low and intermediate fibrosis levels (Bissell 2004, Gastroenterology 2004; 127: 1847-49).
The only reliable and definitive test for liver function and status is a biopsy. However, biopsies cannot be used in standard tests, or for patients with mild conditions or even for routine periodic analysis in patients with severe liver disease. In addition, biopsy has only an 80% accuracy due to the small volume of tissue extracted and variations in the evaluation between pathologists (Poynard et al 2007, BMC Gastroenterol 2007; 7: 40).
The human asialoglycoprotein receptor (ASGPR) is expressed only in hepatocytes and serves in the clearance of asialoglycoproteins from the plasma (Drickamer, K., Cell, 1991. 67(6): p. 1029-1032). ASGPR levels are much lower in developing liver than in fully developed liver. The receptor level is also reduced in patients with cirrhosis and dramatically down-regulated in hepatocarcinomas (Doyle, D. B., Y and Petell, J, ed. The Liver: Biology and Pathobiology., ed. I. J. Arias, W B; Popper, H; Schachter, D and Shafritz, D A. 1988, Raven Press. 141).
The ASGPR is constructed of two subunits of related amino acid sequence, H1 (46 kD) and H2 (50 kD). H2a and H2b are two alternatively spliced variants of the ASGPR H2 subunit (Lederkremer, G. Z. and H. F. Lodish, J Biol Chem, 1991. 266(2): p. 1237-44). H2a differs from H2b only by the presence of an extra pentapeptide in the exoplasmic domain next to the membrane-spanning segment (Lederkremer, G. Z. and H. F. Lodish, J Biol Chem, 1991. 266(2): p. 1237-44). It was shown that H2a is rapidly cleaved next to this pentapeptide to a 35 kDa fragment, comprising the entire ectodomain, which is secreted, constituting a soluble form of the receptor (sH2a). Membrane-bound H2a does not participate in a receptor complex with H1 as is the case for H2b, and thus it is not a subunit of the receptor but a precursor for the soluble secreted form.
Although H2a is a type II transmembrane protein, indirect evidence suggests that signal peptidase is probably responsible for the cleavage to the soluble form. ASGPR sH2a was found to be efficiently secreted from the human hepatoma cell line HepG2. It was discovered that when H2a is expressed in stably transfected NIH 3T3 cells it is also cleaved, however only about 30% of sH2a can be Golgi processed and secreted from transfected fibroblasts and the rest is degraded at the ER (Tolchinsky, S. et al., J. Biol. Chem., 1996, 271(24): p. 14496-14503).
U.S. Application No. 20060286615 teaches diagnosing a liver condition by analyzing a level of sH2A in a sample of a subject.